The binding of Cro and lambda repressor to the synthetic operator DNA has been studied by systematic base substitutions. From this data we deduce specific interactions between Cro and the operator DNA and estimate the energetic contribution of each interaction to the specific binding. Such studies clearly show that the recognition of specific DNA sequences by Cro and lambda repressor is mediated by a combination of bi-dentate H-bonds between amino acid side chains and base pairs, and hydrophobic interactions with thymine's methyl groups as exposed within the DNA major groove. Losses of such H-bond or hydrophobic interactions reduce the binding free energy by 0.9 to 3.1 Kcal/mol or 0.8 to 1.8 Kcal/mol, respectively. The free energy changes are principally additive for the specific binding, but not additive for nonspecific binding. These interactions described here are not only the specificity determinants in the sequence recognition, but also provide a large part of the binding free energy for the specific interactions of Cro and lambda repressor with DNA. Cro and lambda repressor recognize the same operator sequences in quite different ways. Site-directed mutagenesis has been used to further study the interaction between amino acids of cro and base pairs. This study provides valuable information about the nature of the Cro-DNA interaction. Computers have been used to analyze the detailed molecular interactions in the repressor-operator complex, and to assist the site-directed mutagenesis.